insects Article Host Specificity of the Parasitic Wasp Anaphes flavipes (Hymenoptera: Mymaridae) and a New Defence in Its Hosts (Coleoptera: Chrysomelidae: Oulema spp.) Alena Samková 1,2,*, Jiˇrí Hadrava 2,3 , Jiˇrí Skuhrovec 4 and Petr Janšta 2 1 Department of Plant Protection, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, CZ-165 00 Prague 6—Suchdol, Czech Republic 2 Department of Zoology, Faculty of Science, Charles University, Viniˇcná 7, CZ-128 43 Prague 2, Czech Republic; [email protected] (J.H.); [email protected] (P.J.) 3 Institute of Entomology, Biological Centre, Czech Academy of Sciences, Branišovská 31, CZ-370 05 Ceskˇ é Budˇejovice,Czech Republic 4 Group Function of Invertebrate and Plant Biodiversity in Agro-Ecosystems, Crop Research Institute, Drnovská 507, CZ-161 06 Praha 6—Ruzynˇe,Czech Republic; [email protected] * Correspondence: [email protected]; Tel.: +420-607-228-572 Received: 17 January 2020; Accepted: 8 March 2020; Published: 10 March 2020 Abstract: The parasitic wasp Anaphes flavipes (Förster, 1841) (Hymenoptera: Mymaridae) is an important egg parasitoid of cereal leaf beetles. Some species of cereal leaf beetle co-occur in the same localities, but the host specificity of the wasp to these crop pests has not yet been examined in detail. A lack of knowledge of host specificity can have a negative effect on the use of this wasps in biological control programs addressed to specific pest species or genus. In this study, laboratory experiments were conducted to assess the host specificity of A. flavipes for three species of cereal leaf beetles (Oulema duftschmidi Redtenbacher, 1874, Oulema gallaeciana Heyden, 1879 and Oulema melanopus Linnaeus, 1758) in central Europe. For the first time, a new host defence against egg parasitoids occurring in O. gallaeciana from localities in the Czech Republic, a strong dark sticky layer on the egg surface, was found and described. The host specificity of A. flavipes was studied in the locality with the presence of this defence on O. gallaeciana eggs (the dark sticky layer) (Czech Republic) and in a control locality (Germany), where no such host defence was observed. Contrary to the idea that a host defence mechanism can change the host specificity of parasitoids, the wasps from these two localities did not display any differences in that. Respectively, even though it has been observed that eggs with sticky dark layer can prevent parasitization, the overall rate of parasitization of the three species of cereal beetles has not been affected. However, in our view, new host defence can influence the effects of biological control, as eggs of all Oulema spp. in the locality are protected against parasitization from the wasps stuck on the sticky layer of the host eggs of O. gallaeciana. Keywords: parasitoid-host interaction; biological control; host spectrum; Mymaridae 1. Introduction Parasitic wasps occur in high numbers, both in terms of species diversity and absolute numbers of individuals [1]. Despite differing estimates, the diversity of parasitic wasps is assumed to be over one million species, with roughly every tenth species of insects being a parasitic hymenopteran [1,2]. Parasitic wasps attack a wide range of hosts, and they play an important role in the biodiversity and balance of natural ecosystems and agriculture [3,4]. Especially in agriculture, parasitic wasps can be used to reduce important pest insects in biological control programmes [5]. Biological control can be implemented either as modification of landscaping strategies that support natural enemies Insects 2020, 11, 175; doi:10.3390/insects11030175 www.mdpi.com/journal/insects Insects 2020, 11, 175 2 of 11 (conservation biological control) [6] or as releasing (once or repeatedly) of parasitoids or predators in the infested fields or areas (augmentative control) [7,8]. This latter strategy is effective for organic farming and natural protected areas, or if the pests acquire resistance to chemical spraying [9–12]. For example, for augmentative biological control, 170 species of parasitoids are used only in Europe [8]. In some cases, the effectiveness of biological control is up to 100%, e.g., the wasp Cosmocomoidea ashmeadi (Girault, 1915) (Hymenoptera: Mymaridae) controlling the leafhopper Homalodisca coagulata (Say, 1832) (Hemiptera: Cicadellidae) [13], or Anaphes nitens (Girault, 1928) (Hymenoptera: Mymaridae) against the weevil Gonipterus scutellatus Gyllenhal, 1833 (Coleoptera: Curculionidae) [14,15]. Many parasitoids do not have 100% efficiency, and therefore, methods for improving the effectiveness of natural enemies or biological control have been sought [16]. The efficiency of biological control can be increased with detailed knowledge of the host specificity of parasitoids, and host specificity is one of the primary criteria for evaluating the risks of biological control organisms to nontarget organisms [17]. The host range is generally characterized as the set of species on which a control organism can feed and develop [17], all organisms in a given habitat are potentially a host for parasitoids, but their quality and parasitoid approbation is different [1]. The host may be attacked by one to over twenty parasitoids at a time, and the most vulnerable hosts to parasitization appear to be herbivorous insects [18]. Successful parasitization requires the parasitoid first to locate the host’s habitat, then perform a specific behavioral routine to finally lay eggs on or into the host [19]. Parasitoids are also capable of learning novel signals that improve their search efficiency [1]. The hosts are not merely passive participants in this process [3]. They have mechanical, physiological and immune defences against parasitoids and are in a constant evolutionary arms race; whatever defensive mechanisms the host invents, the parasitoid tries to overcome [20,21]. Our study is focused on the host specificity of the potential biological agent, Anaphes flavipes (Föster, 1841) (Hymenoptera: Mymaridae). The host spectrum of this wasp includes the rare Lema spp. and the widespread Oulema spp. (O. duftschmidi Redtenbacher, 1874, O. gallaeciana Heyden, 1879, and O. melanopus Linnaeus, 1758) [22,23]. Larvae and adults of Oulema species damage the leaves of cereals (barley, wheat and oats), and they are an economically important crop pest in Europe and North America [24–26]. For example, in agricultural areas around the world, insect pests reduce grain crop yields by 5% to 20 % every year [27]. The use of parasitic wasps for biological control has been repeatedly tested [28–30]. In this context, the host spectrum of A. flavipes was examined for six taxons, Crioceris duodecimpunctata (Linnaeus, 1758); Oulema sayi (Crotch, 1873); Lema nigrovittata (Guérin-Méneville, 1844); Lema daturaphila Kogan and Goeden, 1970 (as L. trilineata (Olivier, 1808)); Lema trilineata californica (Schaefer Krauss 1947) and Lema trivittata trivittata Say, 1824) by Maltby et al. [31]; however, a current common host species, O. duftschmidi, was not included because, until 1989, it was assigned to O. melanopus [32]. In relation to the study of host specificity of A. flavipes, we describe a new type of host defense against egg parasitoid as a dark sticky layer on host eggs of O. gallaeciana in Czech localities. This sticky layer can completely prevent parasitization, because any females adhere to the sticky layer and are unable to either parasitize or release herself. The eggs defense of cereal beetles against parasitization by parasitoids has been proposed so far only by Anderson and Paschke [22] as a strong selective pressure on the rapid development of beetle larvae. The wasps reject host eggs older than 72 h, probably because the forming larvae could damage the parasitoid egg by sclerotized mandibles. However, there is no experimental evidence for this claim. First, laboratory experiments were carried out to assess the host specificity of Anaphes flavipes for three widespread Oulema species (O. duftschmidi, O. gallaeciana and O. melanopus). Secondly, we described a new type of host defence against parasitoids on eggs and compared the host specificity of the wasp A. flavipes between metapopulations with or without the observed egg defence on three co-occurring Oulema species. The main aim was to test whether the wasp’s choice of host species can be affected by the presence or absence of the host defence. Insects 2020, 11, 175 3 of 11 2. Materials and Methods 2.1. Parasitic Wasps Parasitic wasps (A. flavipes) were collected from host eggs of species O. duftschmidi, O. gallaeciana and O. melanopus in periods from the end of April until the end of June 2015–2016 in cereal fields in one locality in the Czech Republic (50.1385 N, 14.3695 E) and one locality in Germany (50.7787 N, 6.0381 E). The parasitized host eggs were stored in Petri dishes with moistened filter paper until the emergence of the adult wasps. These “wild” wasps were used as an initial population for rearing the next generations of parasitoids in an environmental chamber with conditions of 22 2 C, relative ± ◦ humidity of 40%–60% and 24 h light. All these “next generation” females and males of A. flavipes were bred in laboratory on the eggs Oulema species (O. duftschmidi and O. melanopus) and those used for experiments were at most 24 h old (post emergence). Each emerged female used in the experiment was immediately mated. Each mated female was then placed in a Petri dish with 12 host eggs (8 eggs of O. duftschmidi + O. melanopus, 4 eggs of O. gallaeciana). Before starting and during the experiments, the females were not fed, and they had constant access to water. 2.2. Host Species The host species of the genus Oulema (O. duftschmidi, O. gallaeciana and O. melanopus) were obtained from the adults collected in localities in the Czech Republic (one at the same location as that used for parasitic wasps and one more in Police nad Metují (50.5277 N, 16.2456 E)) and Germany (near the city of Aachen (50.7763 N, 6.0838 E)).